Estradiol derivative-alkylating agent conjugate with reduced hormonal activity, process for preparing the same, compounds useful for the preparation thereof, and growth inhibiting composition containing the conjugate or estradiol derivative

ABSTRACT

Disclosed is an estradiol derivative-alkylating agent conjugate of the formula (I&#39;): ##STR1## wherein R 1  is C 1-4  alkyl or C 1-4  alkoxy; R 2  is acyl or benzyl; m is an integer of 1 to 3; and n is an integer of 0 to 3, which is useful a growth inhibiting agent.

BACKGROUND OF THE INVENTION

The present invention relates to a novel estradiol derivative-alkylatingagent conjugate with reduced hormonal activity, a process for preparingthe conjugate, compounds useful for the preparation of the conjugate,and a growth inhibiting composition containing the conjugate orestradiol derivative.

Among the conventional alkylating agents, although having a strongantitumor activity, there are many agents which fail to display theirfull medicinal effects. The primary reason is their limited dosage dueto their undesirable side effects in medicinal use. A solution to thisproblem is to combine the alkylating agent with a carrier having aspecific affinity for the site of tumor to form an alkylatingagent-carrier conjugate. It is intended to let the alkylating agentaccumulate specifically at the site of tumor so that the agent willexhibit its antitumor activity effectively while suppressing theoccurrence of the undesirable side effects.

Based on this conception, a proposal has been made on preparation of anestradiol-chlorambucil conjugate using estradiol as carrier and anantitumor agent containing the conjugate as principal constituent(Japanese Patent Publication (KOKOKU) No. 58-10397 (1983)). Thisantitumor agent specifically accumulates at the site of tumor andexhibits a strong antitumor activity. Further, it gives almost nosignificant influence to the normal cells.

However, an antitumor agent is usually required to be administered overa long period of time, so that even a slight side effect which is quiteinsignificant in short-term administration may give rise to a seriousproblem in long-time administration of the such agent. Especially,accumulation of the slight side effect may be remarkable as the physicalstrength of the cancer patient is usually weakened.

In the case of the estradiol-chlorambucil conjugate, in long-timeadministration thereof, there is observed in some cases development ofthe same symptoms as seen in administration of estrogen.

The studies by the present inventors have disclosed that development ofsuch symptoms is due to the action of estrogen which is released insmall quantities from the estradiol-chlorambucil conjugate in thepatient's body. It is thus considered that in long-time administrationof this conjugate, estrogen released in small quantities therefrom isaccumulated in some cases to such an extent as to induce its sideeffects.

Further, the present inventors have found that when a specificsubstitutent, for example methyl group, is introduced into the estradiolring, the estrogen action is remarkably diminished without impairing theselective physiological activity (growth-inhibiting action) which isinherently possessed by the estradiol-alkylating agent. It was alsofound that estradiol derivatives usable for the preparation of theconjugate are very low in estrogen action although they have a growthinhibiting activity. The present invention has been attained on thebasis of these findings.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novelestradiol-alkylating agent conjugate which, while maintaining itsoriginal selective physiological activity, is reduced in estrogen actionand of very high utility.

Another object of the present invention is to provide a process forpreparing the conjugate, and estradiol derivatives useful for thepreparation of the conjugate.

Still another object of the present invention is to provide a growthinhibiting agent containing the conjugate or the estradiol derivative,which agent is reduced in estrogen action.

In a first aspect of the present invention, there is provided anestradiol derivative-alkylating agent conjugate of the formula (I'):##STR2## wherein R¹ is C₁₋₄ alkyl or C₁₋₄ alkoxy; R² is acyl or benzyl;m is an integer of 1 to 3; and n is an integer of 0 to 3.

In a second aspect of the present invention, there is provided a processfor preparing the preferred conjugate, which comprises reacting anestradiol derivative of the formula (II): ##STR3## wherein R¹ is C₁₋₄alkyl or C₁₋₄ alkoxy; R² is acyl or benzyl; m is an integer of 1 to 3; nis an integer of 0 to 3; and X is hydroxy or halogen; with a compound ofthe formula (III): ##STR4## wherein R⁴ is carboxy, hydroxy or a saltthereof, halogen, ester group, acid chloride group, acid anhydride groupor methyl halide group; and X is halogen or hydroxy.

In a third aspect of the present invention, there is provided anestradiol derivative of the formula (II): ##STR5## wherein R¹ is C₁₋₄alkyl or C₁₋₄ alkoxy; R² is acyl or benzyl; m is an integer of 1 to 3; nis an integer of 0 to 3; and X is hydroxy or halogen.

In a fourth aspect of the present invention, there is provided a growthinhibiting composition containing an effective amount of the estradiolderivative-alkylating agent conjugate as defined in the first aspect,and a diluent.

In a fifth aspect of the present invention, there is provided a growthinhibiting composition containing an effective amount of an estradiolderivative of the formula (II): ##STR6## wherein R¹ is C₁₋₄ alkyl orC₁₋₄ alkoxy; R² is acyl or benzyl; m is an integer of 1 to 3; n is aninteger of 0 to 3; and X is hydroxy or halogen, and a diluent.

DETAILED DESCRIPTION OF THE INVENTION

The conjugate of the present invention has the structure represented bythe formula (I). In the formula, R¹ is preferably C₁₋₄ alkyl or C₁₋₄alkoxy, more preferably methyl, and exists at the 1- or 4-position or atboth of these positions. Preferably, R² is benzoyl or benzyl.Preferably, m is 2 and n is an integer of 0 to 2. More preferably, n is1 and R¹ is methyl at the 1-position.

At the 3-position of the estradiol derivative moiety, hydrogen of thehydroxy is substituted with acyl or benzyl. Acyl is C₁₋₂₅ acyl, forinstance, benzoyl, acetyl, palmitoyl, stearoyl, linoleoyl or the like.Of these groups, benzoyl is preferred. The configuration at the17-position of the estradiol derivative moiety may be β, α, or a mixturethereof, but β is preferred.

The alkylating agents usable in the present invention are chlorambucil,nitrogen mustard, melphalan and derivatives thereof includingderivatives which have other halogen instead of chlorine. Preferably,the alkylating agent is chlorambucil and a derivative thereof which hasother halogen instead of chlorine. The alkylating agent is bound to anestradiol derivative at a site where the antitumor activity is notimpaired by the binding. Binding between alkylating agent andestradiol-derivative may be effected through the medium of anappropriate spacer.

The preferred conjugate is represented by the formula (I): ##STR7##wherein R¹ R² m and n are as defined above, R³ is carbonyl or methylene,and X is hydroxy or halogen.

Halogen is usually chlorine, bromine or iodine.

The conjugate of the present invention may be prepared, for instance,according to the following process.

First, a synthesis example of an estradiol derivative of the formula(II) is shown in Scheme 1. ##STR8##

The scheme is briefly described below, referring to each of the steps(a) to (g).

Step (a)

A compound of the formula (VI) (which can be prepared according to D. J.Crispin: J. Chem. Soc. (c), 10, 1970, etc.) is reacted with benzylhalide in the presence of sodium hydride at room temperature to form acompound of the formula (V).

Step (b)

The compound of the formula (V) is reacted with a C₁₋₃ straight-chainalkylene glycol in the presence of p-toluenesulfonic acid at 80° to 140°C. to form a compound of the formula (IV).

Step (c)

The compound of the formula (IV) is treated with lithium aluminumhydride in the presence of aluminum chloride at 30° to 70° C. to form acompound of the formula (II-A).

Step (d)

The compound of the formula (II-A) is reacted with tetrahydropyran (THP)at 4° to 60° C. to form a compound of the formula (X).

Step (e)

The compound of the formula (X) is debenzylated by treating it with Pd/Cin H₂ gas at 0° to 30° C. to obtain a compound of the formula (IX).

Step (f)

The compound of the formula (IX) is reacted with an acyl halide at 10°to 30° C. to form a compound of the formula (VIII).

Step (g)

The compound of the formula (VIII) is detetrahydropyranated to give acompound of the formula (II-B).

The compound of the formula (II) may be converted to a salt or halidethereof. A halide can be obtained by treating a compound of the formula(II) wherein X is OH, with p-toluenesulfonyl chloride and sodium halideor the like. A salt can be obtained by the conventional method.

In the estradiol derivatives of the formula (II), preferably R¹ is C₁₋₂alkyl or C₁₋₂ alkoxy, R² is benzoyl or benzyl, m is 2 and n is aninteger of 0 to 2. Most preferably, R¹ is methyl and exists at the 1-and/or 4-position, especially at the 1-position.

Then, the estradiol derivative is combined with the desired alkylatingagent.

In case that the alkylating agent is chlorambucil or a derivativethereof, the estradiol derivative of the formula (II) is reacted with acompound of the formula (III): ##STR9## where R⁴ is carboxy, hydroxy ora salt thereof, halogen, ester group, acid chloride group, acidanhydride group or methyl halide group; and X is halogen or hydroxy, toform a conjugate of the formula (I). The reaction can be carried out inan organic solvent such as dichloromethane, dioxane, dimethyl sulfoxide,dimethylformamide, pyridine, benzene, acetone, toluene, carbontetrachloride, chloroform, tetrahydrofuran or the like. An alkalisolution, sodium hydride, thionyl chloride and the like may be added asdesired The reaction may be performed at -30° to 150° C. preferably -10°to 100° C., for 3 minutes to 48 hours, preferably 5 minutes to 24 hours.The reaction product is purified by a usual method to give the conjugateof the present invention.

The conjugate of the formula (I) in which the 3-position of theestradiol is benzyloxy is debenzylated by treating it with Pd/C in thepresence of H₂ and then further reacted with an acid chloride or acidanhydride corresponding to the objective acyl to provide the conjugateof the present invention in which the 3-position of the estradiol isacyloxy.

Known methods such as extraction, chromatography, crystallization,reprecipitation, etc., can be applied for purification of the reactionproduct.

The toxicity and pharmacological actions, as determined by the followingmethods, of the conjugate and the estradiol derivative of the presentinvention are described below.

(1) Toxicity

The conjugate and the estradiol derivative were each administeredintraperitoneally to the ICR mice at a dose of 100 mg/kg and the testmice were placed under observation for one week. As a result, no deathwas observed.

(2) Estrogen action (uterine weight tests)

The estrogen action of the conjugate and the estradiol derivative wereeach examined according to the uterine weight tests. The estrogen actionwas found diminished.

(3) Growth inhibiting effect (IC₅₀) in testosterone-added system usingbreast cancer cells (SC-115)

Uptake of tritium-labeled thymidine was examined in a serum free culturesystem using testosterone-dependent SC-115 cells. Strong inhibitoryeffects of the conjugate and the estradiol derivatives were shown.

(4) Growth inhibiting effect (IC₅₀) in bFGF-added system using prostaticcancer cells (DC-145)

Uptake of tritium-labeled thymidine was examined in a serum free culturesystem using bFGF-dependent DU-145 cells. Strong inhibitory effects ofthe conjugate and the estradiol derivative were shown.

The conjugate of the formula (I) and the estradiol derivatives of theformula (II) are excessively weakened in estrogen action and low intoxicity and also have a growth inhibiting action, so that they areuseful as a growth inhibiting agent. The substances also show a growthinhibiting action against tumor cells, so that they can be applied as anantitumor agent. They are effective against various types of cancerssuch as breast cancer, ovarian cancer, uterine cancer, prostatic cancer,stomach cancer, rectal cancer, colonic cancer, renal cancer, cancer ofhematopoietic organ, liver cancer, cancer of urinary organ, and othersolid cancers. The conjugate and the estradiol derivative also have aninhibiting action against prostatic hypertrophy and are therefore usefulas an anti-prostatic hypertrophy agent.

For use of the conjugate and the estradiol derivative as a growthinhibiting agent, it can be prepared into various forms suited for theways of administration in combination with a diluent (carrier) accordingto the known methods. In case of preparation of a pharmaceuticalcomposition, a pharmaceutically acceptable diluent is used. The forms ofcomposition include capsule, syrup, oral preparations such as pills andtablets, injections, external preparations, suppository, etc. Theexternal preparations include solid agents containing a percutaneouspenetration auxiliary such as lauric acid diethanol amide in an ordinarybase such as white vaseline.

In the composition, the conjugate and the estradiol derivative each maybe contained in an amount of preferably 0.01 to 75% by weight, morepreferably 0.05 to 25% by weight. The conjugate and the estradiolderivative can be administered through various routes such as peroral,percutaneous, intramuscular, intravenous, intraarterial, intrarectal,etc. The dosage is variable depending on the way of administration andthe degree of treatment, but generally it is 0.1 to 50 mg/kg per day inthe case of peroral administration and 0.01 to 20 mg/kg per day in thecase of parenteral administration, both for adults.

The estradiol derivative moiety in the conjugate of the presentinvention has an ether linkage at the 17-position in addition to asubstitutent at the 3-position of the A ring. This structure isconsidered to lend itself to excessive diminution of the estrogen actionwhile allowing maintenance of the growth inhibiting action which isinherently possessed by the estradiol-alkylating agent conjugate, thusremarkably enhancing the utility of the conjugate as a growth inhibitingagent.

EXAMPLES

The following examples further illustrate the present invention. Theseexamples are merely intended to be illustrative and not to be taken aslimiting the scope of the invention. "%" is by weight, unless otherwisenoted. The properties of the compounds were determined by using thefollowing material or apparatus.

(1) Thin layer chromatography (silica gel): Merck's Kieselgel 60 F₂₅₄

(2) Elemental analysis: Yanagimoto C. H. N. Coder MT-3 (Yanagimoto Ltd.)

(3) Mass spectrometry: Mass Spectrometer JMS-DX 303 (Nippon Denshi KK)

(4) NMR (CDCl₃): JNM-GSX-500 (Nippon Denshi KK)

(5) Infrared absorption spectrometry: Infrared Spectrophotometer A-202(Nippon Bunko KK)

Example 1 Synthesis of 3-benzyloxy-1,3,5(10) -estratriene-17-one (V-1)

3-Hydroxy-1,3, 5 (10) -estratriene-17-one (VI-1) (2.70 g), distilledtetrahydrofuran (THF) (20 ml) and dimethylformamide (DMF) (4 ml) weremixed and dissolved in an eggplant type flask (300 ml). To this solutionwere added sodium hydride (0.24 g) and benzyl bromide (1.88 g), and themixture was stirred overnight at room temperature. Distilled water wasadded to the reaction solution, and the solution was extracted withethyl acetate (100 ml), followed by additional extraction with ethylacetate (50 ml). The organic layers were washed with distilled water anda saturated saline solution. The washings were dried over anhydroussodium sulfate and concentrated under reduced pressure to give theobjective compound (V-1) (3.48 g) as crystals.

Yield: 96.5%

Elemental analysis, calcd. for C₂₅ H₂₈ O₂ :

Found: C, 82.65; H, 7.82.

Calcd.: C, 83.2 9; H, 7.83.

Melting point: 129.2°-130.8° C.

¹ H-NMR (CDCl₃, δppm): 0.91 (s, 3H), 1.39-1.66 (m, 8H), 1.94-2.08 (m,3H), 2.12-2.17 (m, 1H), 2.25 (m, 1H), 2.37 (m, 1H), 2.49 (m, 1H), 2.89(m, 2H), 5.04 (s, 2H), 6.73 (s, 1H), 6.79 (dd, 1H), 7.20 (d, 1H), 7.31(t, 1H), 7.38 (t, 2H), 7.42 (d, 2H).

IR (KBr, νmax): 3460 m, 3080 m, 3050 m, 2940 s, 2980 s, 1738 s, 1605 s,1580 m, 1510 s.

Rf: 0.46 (hexane/ethyl acetate=2:1)

Example 2 Synthesis of 3-benzyloxy-1,3,5(10)-estratriene-17-cycloethylene ketal (IV-1)

Benzene was added to 3-benzyloxy-1,3,5 (10) -estratriene-17-one (V-1)(0.11 g) , ethylene glycol (0.23 g) and p-toluenesulfonic acid (0.6 mg).This mixture was azeotropically boiled at 120° C. by a Dean-Starkapparatus and stirred overnight. An excess amount of triethylamine wasadded to the reaction solution to neutralize, and the mixed solution wasconcentrated under reduced pressure and extracted with ethyl acetate (50ml). The organic layer was washed with distilled water, dried overanhydrous sodium sulfate and concentrated under reduced pressure toproduce the crystals (0.14 g). These crystals were purified by silicagel chromatography (hexane/ethyl acetate=4:1) to give the objectivecompound (IV-1) (0.11 g) as white crystals.

Yield: 94.0%.

Elemental analysis, calcd. for C₂₇ H₃₂ O₃ :

Found: C, 79.81; H, 7.96.

Calcd.: C, 80.16; H, 7.97.

Melting point: 124.5°-125.5° C.

¹ H-NMR (CDCl₃, δppm): 0.88 (s, 3H), 1.32-1.57 (m, 6H), 1.64 (m, 1H),1.74-1.91 (m, 4H), 2.03 (m, 1H), 2.24 (m, 1H), 2.31 (m, 1H), 2.84 (m,2H), 5.03 (s, 2H), 3.87-3.98 (m, 4H), 6.71 (s, 1H), 6.77 (dd, 1H), 7.20(d, 1H), 7.30 (m, 1H), 7.37 (t, 2H), 7.42 (d, 2H).

IR (KBr, νmax): 3460 m, 3100 m, 3070 m, 3020 s, 2950 s, 2910 s, 2850 s,1610 s, 1590 m.

Rf: 0.56 (hexane/ethyl acetate=2:1)

Example 3 Synthesis of 2-[3-benzyloxy-1,3,5 (10)-estratriene-17β-oxy]ethanol (II-1)

Aluminum chloride (14.9 mmol) and anhydrous ether (4 ml) were placedinto an eggplant type flask (100 ml) at 0° C., and the solution wasstirred for 30 minutes. To this solution was added a suspension oflithium aluminum hydride (3.6 mmol) and anhydrous ether (4 ml), and themixture was stirred for additional 30 minutes. To this solution wasadded an anhydrous ether (20 ml) solution of 3-benzyloxy-1,3,5(10)-estratriene-17-cycloethylene ketal (IV-1) (3:6 mmol), and the mixedsolution was refluxed under heating at about 50° C. for 3 hours,followed by addition of distilled water at 0° C. This solution wastransferred into an eggplant type flask (500 ml) and the solute wasextracted with diethyl ether (200 ml). The organic layer was dried overanhydrous sodium sulfate and concentrated under reduced pressure toproduce the crystals (1.34 g) . These crystals Were washed with hexaneto give the objective compound (II-1) (1.20 g) as white crystals.

Yield: 81.0%.

Elemental analysis, calcd. for C₂₇ H₃₂ O₃ :

Found: C, 79.43; H, 8.43.

Calcd.: C, 79.77; H, 8.43.

Melting point: 82.8°-84.3° C.

¹ H-NMR (CDCl₃, δppm): 0.80 (s, 3H), 1.20 (m, 1H), 1.30-1.57 (m, 7H),1.68 (m, 1H), 1.68 (m, 1H), 2.01-2.07 (m, 5H), 2.23 (m, 1H), 2.83 (m,2H), 3.43 (t, 1H), 3.56 (m, 1H), 3.61 (m, 1H), 3.70 (m, 2H), 5.02 (s,2H), 6.70 (s, 1H), 6.77 (dd, 1H), 7.19 (d, 1H), 7.30 (m, 1H), 7.37 (t,2H), 7.42 (d, 2H).

IR (KBr, νmax): 3460 s, 3100 m, 3260 m, 2950 s, 2900 s, 1610 s, 1590 m,1515 s.

Rf: 0.22 (hexane/ethyl acetate=2:1)

Example 4 Synthesis of 2-[3-benzoyloxy-1,3,5(10)-estratriene-1-methyl-17β-oxy]ethanol (II-3)

2-[3-benzyloxy-1,3,5(10) -estratriene-1-methyl-17β-oxy]ethanol (II-2)(m.p.: 103.5°-104.5° C.) (2.31 g) produced according to the process ofExample 3 and dichloromethane (15 ml) were placed and dissolved in aneggplant type flask (100 ml). To this solution were added dihydropyran(924 mg) and pyridinium p-toluenesulfonate (PPTS) (138 mg), and themixture was stirred overnight at room temperature. The reaction solutionwas distilled, and the residue was dissolved in ethyl acetate (50 ml),washed twice with water (15 ml) and further washed with a saturatedsaline solution (15 ml). The washings were dried over Na₂ SO₄, filteredand distilled to form an oily substance (3.19 g) . This substance waspurified by silica gel chromatography (hexane/ethyl acetate=5:1) to givea purified product (2.42 g). The progress of the reaction was confirmedby TLC check. The purified product was dissolved in dioxane (238 ml) and10% Pd/C was added to the solution. This solution was treated with H₂gas at room temperature for 5 hours to obtain a debenzylated compound(1.77 g). This compound was dissolved in dry dichloromethane (30 ml),followed by addition of pyridine (8.5 ml) and benzoyl chloride (596.0ml), and the mixed solution was stirred overnight at room temperature.The stirred solution was distilled and the residue was dissolved inethyl acetate (60 ml), washed twice with water (20 ml) and furtherwashed with a saturated saline solution (20 ml). The washings weredistilled and the residue was crystallized. The crystallized product wasfiltered out, collected and washed with hexane (3 ml) to produce abenzoyl compound (1.53 g). This compound was dissolved in ethanol (85ml) and PPTS (1.24 g), and the solution was stirred at 40° C. for 2hours and distilled. The residue was dissolved in ethyl acetate (100 ml)and washed with water (30 ml) and a hydrochloric acid solution (1N, 30ml). The organic layer was dried over Na₂ SO₄, filtered and distilled.The residue was crystallized, again dissolved in ethyl acetate/hexane(1:3) (50 ml) and recrystallized. The produced crystals were filteredout, washed with hexane (20 ml) and dried in vacuo to give the objectivecompound (II-3) (1.07 g) as white needle crystals.

Melting point: 185°-187° C.

¹ H-NMR (CDCl₃, δppm): 0.80 (s, 3H), 1.24 (m, 2H), 1.37 (m, 2H), 1.45(m, 2H), 1.67 (m, 1H), 1.80 (m, 2H), 2.00 (m, 1H), 2.23 (s, 3H), 2.37(t, 1H), 2.65 (m, 2H), 2.72 (m, 1H), 3.35 (t, 1H), 3.50 (m, 2H), 3.64(m, 2H), 6.84 (d, 1H), 7.04 (d, 1H), 7.51 (m, 2H), 7.64 (m, 1H), 8.18(m, 2H).

IR (KBr, νmax): 3500 br, 2950 m, 2880 m, 1735 s, 1600 w, 1580 w, 1450 m,1380 w, 1340 w, 1320 w, 1265 s, 1220 s, 1160 m, 1100 m, 1085 s.

Mass spectrum (m/e): 434 (M+), 416, 312, 268, 251, 227.

Example 5 Synthesis of 1-[3-benzyloxy-1,3,5(10)-estratriene-17β-oxy]-2-[4-[p-[bis(2-chloroethyl)amino]phenyl]butoxy]ethane (I1)

2-[3-benzyloxy-1,3,5(10) -estratriene-17β-oxy]ethanol (II-1) (61 mg) anddistilled DMF (0.5 ml) were placed and dissolved in an eggplant typeflask (200 ml). To this solution was added sodium hydride (5 mg) at 0°C., followed by further addition of4-[p-[bis(2-chloroethyl)amino]phenyl]-iodobutane (Rf=0.64,phosphomolybdic acid reagent) (90 mg) at 0° C. and the mixture wasstirred for 3 hours. Distilled water was added to the reaction solution,and the solution was extracted twice with ethyl acetate (50 ml). Theorganic layers were washed with a saturated saline solution (50 ml), andthe washings were dried over anhydrous sodium sulfate and concentratedunder reduced pressure to give an oily substance (0.19 g). This oilysubstance was purified by silica gel chromatography (hexane/ethylacetate=6:1) to give the objective compound (I-1) (56.1 mg) as an oilysubstance.

Yield: 55.1%.

¹ H-NMR (CDCl₃, δppm): 0.79 (s, 3H), 1.19 (m, 1H), 1.29-1.58 (m, 10H),1.63 (m, 4H), 1.86 (m, 1H), 2.03 (m, 2H), 2.15 (m, 1H), 2.25 (m, 1H),2.54 (t, 2H), 2.83 (m, 2H), 3.43 (t, 1H), 3.50 (t, 2H), 3.55 (t, 2H),3.59 (s, 5H), 3.66 (m, 5H), 5.03 (s, 2H), 6.61 (d, 2H), 6.70 (d, 1H),6.77 (dd, 1H), 7.06 (d, 2H), 7.18 (d, 1H), 7.31 (t, 1H), 7.37 (t, 2H),7.42 (d, 2H).

IR (KBr, νmax): 2950 s, 2870 s, 1638 s, 1575 m, 1525 s, 1510 s, 1455 m.

Rf: 0.58 (hexane/ethyl acetate=2: 1).

Example 6 Synthesis of1-[3-benzoyloxy-1,3,5(10)-estratriene-17β-oxy]-2-[4-[p-[bis(2-chloroethyl)amino]phenyl]butoxy]ethane (I2)

10% palladium on carbon (83.1 mg) was added at 0° C. into an eggplanttype flask (100 ml), followed by piecemeal addition of 1,4-dioxane (3ml). To this solution was added a 1,4-dioxane (5 ml) solution of1-[3-benzyloxy-1,3,5(10)-estratriene-17δ-oxy]-2-[4-[p-[bis(2-chloroethyl)amino]-phenyl]butoxy]ethane (I-1) (0.11 g)piecemeal at 0° C. The mixture was heated to room temperature andstirred under hydrogen gas for 2 hours. This reaction mixture wasfiltered through filter paper while washing with 1,4-dioxane, and thefiltrate was concentrated under reduced pressure to produce an oilysubstance (92.7 mg). This oily substance was dissolved indichloromethane (0.5 ml), followed by addition of pyridine (0.3 ml) andbenzoyl chloride (0.02 ml) at 0° C., and stirred overnight at roomtemperature. The reaction solution was extracted with ethyl acetate (50ml) and the extract was washed with a saturated saline solution. Thewashings were dried over anhydrous sodium sulfate and concentrated underreduced pressure to produce an oily substance (116.7 mg). It waspurified by silica gel chromatography (hexane/ethyl acetate=4: 1) togive the objective compound (I-2) (74.1 mg) as white crystals.

Melting point: 96.8°-98.7° C.

Mass spectrum (m/e): 705 (M⁺)

Rf: 0.54 (hexane/ethyl acetate=2: 1)

In the same way,1-[3-benzoyloxy-1,3,5(10)-estratriene-1-methyl-17β-oxy]-2-[4-[p-[bis(2-hydroxyethyl)amino]phenyl]-butoxy]ethane(I-3) was synthesized.

Rf: 0.36 (n-hexane/ethyl acetate=2:1)

Mass spectrum (m/e):705 (M+), 656, 371, 224, 105.

IR: 3095 w, 2950 s, 2880 s, 1740 s, 1622 s, 1525 s, 1460 s, 1396 m, 1360s.

There was also similarly synthesized1-[3-benzoyloxy-1,3,5(10)-estratriene-17β-oxy]-2-]4-]bis(2-hydroxyethyl)-amino]-phenyl]butoxy]ethane(I-4).

Example 7 Synthesis of1-[3-benzyloxy-1,3,5(10)-estratriene-17β-oxy]-2-[4-[p-[bis(2-chloroethyl)amino]phenyl]-butyryloxy]ethane(I-5)

2-[3-benzyloxy-1,3,5(10)-estratriene-17β-oxy]ethanol (II-1) anddichloromethane (5 ml) were placed and dissolved in an eggplant typeflask (300 ml), followed by addition of pyridine (95 ml) at 0° C. Tothis solution, chlorambucil (0.3 g) and excess thionyl chloride wereadded dropwise and stirred at room temperature for about 5 minutes.Benzene was added to this reaction solution, and the solution wasconcentrated under reduced pressure three times. To the residue wasadded a small quantity of dichloromethane while washing therewith at 0°C. and the mixture was stirred overnight. The reaction mixture waspoured into cold water, and the mixture was extracted twice with diethylether (50 ml). The organic layers were dried over anhydrous sodiumsulfate and concentrated under reduced pressure to produce an oilysubstance (0.72 g). It was purified by silica gel chromatography(hexane/ethyl acetate=4:1) to give the objective compound (I-5) (0.49g).

Yield: 71.1%.

¹ H-NMR (CDCl₃, δppm): 0.78 (s, 3H), 1.18 (m, 1H), 1.29-1.58 (m, 6H),1.67 (m, 1H), 1.87 (m, 1H), 1.92 (d, 2H), 2.18 (m, 1H), 2.25 (m, 1H),2.35 (t, 2H), 2.5 (t, 2H), 2.84 (m, 2H), 3.42 (t, 1H), 3.60 (m, 5H),3.67 (m, 5H), 4.21 (t, 2H), 5.03 (s, 2H), 6.64 (d, 2H), 6.71 (s, 1H),6.77 (dd, 1H), 7.07 (d, 1H), 7.18 (d, 1H), 7.31 (t, 1H), 7.37 (t, 2H),7.42 (d, 2H).

IR (KBr, νmax): 3460 m, 3080 m, 3050 m, 2940 s, 2980 s, 1738 s, 1605 s,1580 m, 1510 s.

Rf: 0.54 (hexane/ethyl acetate=2: 1)

Mass spectrum (m/e): 691 (M+), 637.

Example 8 Synthesis of1-[3-benzoyloxy-1,3,5(10)-estratriene-17β-oxy]-2-[4-[p-[bis(2-chloroethyl)amino]phenyl]-butyryloxy]ethane (I-6)

10% palladium on carbon (320.7 mg) was placed into an eggplant typeflask (100 ml), followed by dropwise addition of 1,4-dioxane (15 ml) at0° C. To this mixture was added a 1,4-dioxane (15 ml) solution of1-[3-benzyloxy-1,3,5(10) -estratriene-17β-oxy]-2-[4-[p-[bis(2-chloroethyl)amino]-phenyl]-butyryloxy]ethane (I-5) dropwise at 0° C.The mixture was heated to room temperature and stirred under hydrogengas for one hour. The reaction mixture was filtered through filter paperwhile washing with 1,4-dioxane, and the filtrate was concentrated underreduced pressure to produce an oily substance (0.27 g). It was purifiedby silica gel chromatography (hexane/ethyl acetate=3:1) to obtain1-[3-hydroxy-1,3,5(10)-estratriene-17β-oxy]-2-[4-[p-[bis(2-chloroethyl)-amino]phenyl]butyryloxy]ethane (0.26 g) (Rf: 0.25,hexane/ethyl acetate=3:1) as oily substance.

To a 1,4-dioxane (1 ml) solution of this oily substance, pyridine (0.8ml) and benzoyl chloride (0.1 ml) were added at 0° C. and stirred atroom temperature for 2.5 hours. The reaction solution was extracted withethyl acetate (50 ml) and the extract was washed with a saturated salinesolution. The washings were dried over anhydrous sodium sulfate andconcentrated under reduced pressure to produce an oily substance (0.36g). This oily substance was purified by silica gel chromatography(hexane/ethyl acetate=4:1) to give the objective compound (I-6) (0.12 g)as white crystals.

Yield: 71.1%.

Elemental analysis, calcd. for C₄₁ H₄₉ O₅ NCl₂ :

Found: C, 68.87; H, 6.84; N, 2.12.

Calcd.: C, 69.68; H, 6.99; N, 1.98.

Melting point: 113.7°-115.0° C.

¹ H-NMR (CDCl₃, δppm): 0.80 (s, 3H), 1.35-1.57 (m, 8H), 1.93 (m, 2H),2.21 (m, 2H), 2.36 (t, 2H), 2.58 (t, 2H), 2.88 (m, 2H), 3.43 (t, 1H),3.61 (m, 5H), 3.68 (m, 5H), 4.21 (t, 2H), 6.65 (d, 2H), 6.92 (s, 1H),6.97 (d, 1H), 7.08 (d, 2H), 7.32 (d, 1H), 7.50 (t, 2H), 7.63 (t, 1H),8.19 (d, 2H).

IR (KBr, νmax): 2990 m, 2950 m, 2900 m, 1755 s, 1735 s, 1620 m, 1610 m,1530 m, 1515 m.

Rf: 0.48 (hexane/ethyl acetate=2:1).

Mass spectrum (m/e): 105 (Ph-CO), 128 (2CH₂ CH₂ Cl), 656 (M(-CH₂ Cl)),705 (M+).

Example 9 Toxicity test

The compounds of the present invention dissolved in sesame oil(containing 3% benzyl alcohol) were each administered onceintraperitoneally to 6-week-old ICR female mice (average body weight: 25g) at a dosage of 100 mg/kg. The test mice were observed for a period of7 days after administration of the compounds. No death was observed inany case of the compounds.

Example 10 Estrogen action (uterine weight tests)

The test compounds and estradiol dissolved in sesame oil (containing 3%benzyl alcohol) were each administered once hypodermically to 3-week-oldWistar female rats. 24 hours later, the uterus of each test rat wasremoved and its weight (ratio to the body weight) was measured. From therelation between the uterus weight/body weight ratio and the dosage, thecompound dosage that gives a uterus weight/body weight ratio twice thatin the control group was determined. The dosages of the test compoundsare shown as a ratio to the dosage of estradiol.3-Benzoyloxy-1,3,5(10)-estratriene-17β-[4-[p-[bis(2-chloroethyl)amino]phenyl]-butyryloxy]acetate was used as acomparative compound. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                        Dosage that gives a uterus                                                    wt/body wt ratio twice that                                   Test            in control group                                              compound        (ratio to estradiol dosage)                                   ______________________________________                                        II-1             200                                                          II-2            >7000                                                         II-3            >7000                                                         I-1             >7000                                                         I-2             >7000                                                         I-3             >7000                                                         I-4              1400                                                         I-5             >7000                                                         I-6             >7000                                                         Comparative      200                                                          compound                                                                      ______________________________________                                    

Example 11 Growth inhibiting effect (IC₅₀) in testosterone-added systemusing SC-115 cells

The SC-115 cells were cultured on a 96-well microplate (5,000cells/well) for 24 hours with a culture medium (200 μl/well; Ham'sF-12/MEM-E (98% ), dextran-treated fetal calf serum (2%) andtestosterone (1×10⁻⁸ M)). The culture medium was replaced with freshmedium containing the test compound (Ham's F-12/MEM-E, bovine serumalbumin (0.1%), testosterone (1×10⁻⁸ M) and test compound of variousconcentrations). The culture medium was replaced every two days, andculture was continued for 7 days. Thymidine (1 μci/ml, tritium-labeled)was added, and 5 hours later, the culture medium was removed. The cellswere washed with PBS and then washed twice with ice-cold trichloroaceticacid (10%). Then the cells were dissolved in 0.5N sodium hydroxide andneutralized with 0.5N hydrochloric acid. The radioactivity of theneutralized solution was measured by a liquid scintillation counter todetermine the growth inhibiting effect (IC50). The results are shown inTable 2.

                  TABLE 2                                                         ______________________________________                                        Test compound   IC.sub.50  (μM)                                            ______________________________________                                        II-1            0.2                                                           II-2            0.7                                                           II-3            1.5                                                           I-1             1.2                                                           I-2             1.3                                                           I-3             0.2                                                           I-4             0.8                                                           I-5             0.5                                                           I-6             1.5                                                           Comparative     2.6                                                           Compound                                                                      ______________________________________                                    

Example 12 Growth inhibiting effect (IC₅₀) in bFGF-added system usingDU-145 cells

The DU-145 cells were cultured on a 96-well microplate (5,000cells/well) for 24 hours with a culture medium (200 μl/well; Ham'sF-12/MEM-E (98%) and dextran-treated fetal calf serum (2%)). Then theculture medium was replaced with fresh medium containing the testcompound (Ham's F-12/MEM-E, bovine serum albumin (0.1%), bovinepituitary-derived bFGF (1 ng/ml) and the test compound of variousconcentrations). Culture with this medium was continued for 4 days.Thymidine (1 μci/ml, tritium-labeled) was added, and 5 hours layer, theculture medium was removed. The cells were washed with PBS and thenwashed twice with ice-cold trichloroacetic acid (10%). Then the cellswere dissolved in 0.5N sodium hydroxide and neutralized with 0.5Nhydrochloric acid. The radioactivity of the neutralized solution wasmeasured by a liquid scintillation counter to determine the growthinhibiting effect (IC₅₀). The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Test compound   IC.sub.50  (μM)                                            ______________________________________                                        II-1            2.9                                                           I-1             4.6                                                           I-2             7.8                                                           I-3             9.2                                                           Comparative     ND*                                                           Compound                                                                      ______________________________________                                         *Under the experimental conditions, no inhibiting action against growth o     the cells was observed at the concentrations (0.1 to 10 μM) in the         range of measurement.                                                    

Example 13

    ______________________________________                                                                  (by                                                 Preparation example       weight)                                             ______________________________________                                        Compound of the present invention (I-1)                                                                 30 parts                                            Mannitol                  35 parts                                            Sorbitol                  25 parts                                            Carboxymethyl cellulose   5 parts                                             Magnesium stearate        5 parts                                             Talc                      40 parts                                            ______________________________________                                    

What is claimed is:
 1. An estradiol derivative-alkylating agentconjugate of the formula (I'): ##STR10## wherein R¹ is C₁₋₄ alkyl orC₁₋₄ alkoxy; R² is acyl or benzyl; m is an integer of 1 to 3; and n isan integer of 0 to
 3. 2. A conjugate according to claim 1, whereinconfiguration at 17-position of the estradiol derivative moiety is β. 3.A conjugate according to claim 1, wherein R² is benzyl or benzoyl.
 4. Aconjugate according to claim 1, wherein n is 1 and R¹ is methyl at1-position.
 5. A conjugate according to claim 1, represented by theformula (I): ##STR11## wherein R¹ R² m and n are as defined in claim 1;R³ is carbonyl or methylene; and X is hydroxy or halogen.
 6. A growthinhibiting composition comprising an effective amount of the estradiolderivative-alkylating agent conjugate as defined in claim 1, and adiluent.